Conserved motif of CDK5RAP2 mediates its localization to centrosomes and the Golgi complex

Abstract

As the primary microtubule-organizing centers, centrosomes require gamma-tubulin for microtubule nucleation and organization. Located in close vicinity to centrosomes, the Golgi complex is another microtubule-organizing organelle in interphase cells. CDK5RAP2 is a gamma-tubulin complex-binding protein and functions in gamma-tubulin attachment to centrosomes. In this study, we find that CDK5RAP2 localizes to the Golgi complex in an ATP- and centrosome-dependent manner and associates with Golgi membranes independently of microtubules. CDK5RAP2 contains a centrosome-targeting domain with its core region highly homologous to the Motif 2 (CM2) of centrosomin, a functionally related protein in Drosophila. This sequence, referred to as the CM2-like motif, is also conserved in related proteins in chicken and zebrafish. Therefore, CDK5RAP2 may undertake a conserved mechanism for centrosomal localization. Using a mutational approach, we demonstrate that the CM2-like motif plays a crucial role in the centrosomal and Golgi localization of CDK5RAP2. Furthermore, the CM2-like motif is essential for the association of the centrosome-targeting domain to pericentrin and AKAP450. The binding with pericentrin is required for the centrosomal and Golgi localization of CDK5RAP2, whereas the binding with AKAP450 is required for the Golgi localization. Although the CM2-like motif possesses the activity of Ca(2+)-independent calmodulin binding, binding of calmodulin to this sequence is dispensable for centrosomal and Golgi association. Altogether, CDK5RAP2 may represent a novel mechanism for centrosomal and Golgi localization.

FIGURE 1.

Golgi localization of CDK5RAP2. A, HeLa cells were stained with the monoclonal CDK5RAP2 antibody and other antibodies as labeled. Mann II, mannosidase II. Cell cycle stages were identified by the centrin patterns (insets). B–D, cells were subjected to treatment with brefeldin A (B), nocodazole (C), or 2-deoxy-d-glucose and sodium azide (D). Scale bars, 5 μm.

FIGURE 2.

Centrosomes are required for the localization of CDK5RAP2 to the Golgi complex. HeLa cells expressing centrin-GFP were subjected to laser ablation of the centrosome or a cytoplasmic area (inside the boxed areas). Enlarged is the centrosomal area before and after ablation. After ablation, cells were cultured for 2 h and then processed for immunostaining. Shown are the representatives of five centrosome-ablated or control ablation cells from three separate experiments. Scale bar, 5 μm.

FIGURE 3.

Mapping the Golgi-targeting region. Cells transfected with CDK5RAP2 fragments or mutant (FLAG-tagged) were processed for anti-FLAG and anti-GM130 staining. 1456–1893(ΔCBD), 1456–1893 deleted from fragment(1861–1870). Representative images of three independent experiments are presented. Scale bar, 5 μm.

FIGURE 4.

CDK5RAP2 contains a conserved centrosome-targeting domain adjacent to the C terminus. A, sequence alignment of a C-terminal region from CDK5RAP2 and related proteins. Two putative proteins are from chicken (Gallus gallus; GenBank accession no. XP_415517) and zebrafish (Danio rerio; GenBank accession no. CAI11891). Sequence in red is the predicted CaM-binding motif. Asterisks mark Lys¹⁸⁶⁵ and Lys¹⁸⁶⁹ that are mutated for CaM-binding tests. B, C-terminal fragments of CDK5RAP2 transiently expressed in fusion with GFP at the N terminus. The cells were stained with a centrin antibody. Arrows denote centrosomes. Fluorescent intensities were determined at different expression levels of the proteins to derive the intensity ratios of the centrosomes to the cytoplasm. The cytoplasmic GFP signals are expressed in arbitrary units (A.U.). C, HeLa cells transfected with GFP-tagged 1726–1893 (upper) and stained for Odf2/hCenexin to identify mother centrioles. Untransfected cells (lower) were stained for endogenous CDK5RAP2 and Odf2/hCenexin. The same results were obtained in three independent experiments. Scale bars, 5 μm.

FIGURE 5.

CaM binds to the CM2-like motif of CDK5RAP2. A, bacterial extracts expressing His₆-tagged 1660–1893 were prepared in the presence of either 2 mm Ca²⁺ or 5 mm EGTA. After incubation of CaM-conjugated or blank Sepharose in the extracts, proteins bound to the beads were immunoblotted with an anti-His₆ antibody. B, fragment(1660–1893) and its mutants were transiently expressed in HEK293T with a GFP tag. The cells were extracted either in the Ca²⁺-containing or in the EGTA-containing buffer. After binding of proteins in the extracts to CaM-conjugated or blank Sepharose, bound proteins and the inputs were analyzed on anti-GFP immunoblots. ΔCBD, 1660–1893(Δ1861–1870); K1865/9A, 1660–1893(K1865A/K1869A); WT, wild type. C, fragment(1726–1893) and its ΔCBD and K1865A/K1869A mutants were expressed in HeLa cells as GFP-tagged proteins. The cells were subjected to anti-centrin immunostaining. The same results were obtained in three independent experiments. Scale bar, 5 μm.

FIGURE 6.

Centrosomal and Golgi localization of CDK5RAP2 and its mutants. A, CDK5RAP2 wild-type (WT) and the ΔCBD and K1865A/K1869A mutants expressed in HeLa cells. The cells were stained for FLAG-CDK5RAP2 (anti-FLAG), centrioles (GT335), TGN46, and DNA. Boxed areas are enlarged. ΔCBD, CDK5RAP2(ΔCBD); K1865/9A, CDK5RAP2(K1865A/K1869A). B, fluorescent intensity ratios of the centrosomes to the cytoplasm. Analyzed are cells expressing the wild type or the mutants at various levels. Representative results of three independent experiments for each panel are shown. Scale bar, 5 μm.

FIGURE 7.

CM2-like motif is required for binding of several PCM/Golgi proteins to the C-terminal region of CDK5RAP5. A, HeLa cells transfected with FLAG-(1726–1893) or its ΔCBD mutant were subjected to anti-FLAG immunoprecipitation. The immunoprecipitates (IPs) and the extracts (Inputs) were analyzed on immunoblots. ΔCBD, fragment (1726–1893)(ΔCBD). B, cells transfected with control or pericentrin-targeting siRNA immunostained for CDK5RAP2, centrin2, and TGN46. C, HeLa cells transfected with control or AKAP450-targeting siRNA subjected to immunostaining. Data shown are representative from three independent experiments. Scale bars, 5 μm.

FIGURE 8.

Overexpression of the C-terminal fragments delocalizes endogenous CDK5RAP2 from centrosomes and the Golgi complex. A, HeLa cells were transfected with the CDK5RAP2 fragment(1726–1893) or its ΔCBD mutant. The cells were stained for endogenous CDK5RAP2 and mannosidase II (Mann II). WT, wild type; ΔCBD, fragment(1861–1870) deletion mutant. B, cells expressing fragment(1726–1893) were stained for γ-tubulin. The centrosomal area is enlarged from an untransfected (solid line) and a transfected (dashed line) cell. C, cells were transfected with fragment(1456–1893) and then stained for endogenous CDK5RAP2 and TGN46. Representative data of three separate experiments for each panel are presented. Scale bars, 5 μm.